Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery
Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery
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DOI：
10.1038/sj.gt.3300708
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发表时间：
1998-09-01
期刊：
影响因子：
5.1
通讯作者：
Jans, DA
中科院分区：
文献类型：
--
作者：
Chan, CK;Hubner, S;Jans, DA
一种新的非病毒DNA递送方法是使用DNA结合蛋白如酵母转录激活因子GAL 4和质粒DNA的组合，所述质粒DNA含有插入其中的DNA结合蛋白的特异性结合序列，以及待转移到靶细胞中的目的基因。GAL 4的氨基末端147个氨基酸含有DNA结合结构域，其已显示特异性结合17 bp核苷酸识别序列，而氨基末端74个氨基酸已显示足以将大的异源蛋白靶向细胞核。虽然以前已经被用作基因转移载体，但GAL 4的DNA结合和核靶向活性之间的确切关系尚未研究。本研究使用凝胶迁移率变化测定和基于ELISA的结合测定，直接研究了这个问题，建立了GAL 4的DNA结合和核靶向活性的相互排他性。我们证明GAL 4（1-147）可以特异性地增强含有17 bp识别序列的质粒的转染。有趣的是，我们发现GAL 4的核定位信号（NLS）与常规NLS如SV 40大肿瘤抗原和二分NLS不同，因为它只被NLS-受体输入蛋白复合物的核孔靶向β-亚基而不是α-亚基识别。β-亚基结合阻断了与DNA的特异性结合，匡威亦然，这使得GAL 4-NLS受到DNA结合的调节，这可能在影响GAL 4通过核孔转运后从β-亚基单位释放方面发挥重要作用。这项研究首次直接分析了NLS在非病毒DNA转移载体中的识别/可及性，结果与GAL 4和类似的DNA结合蛋白在这种载体中的使用有关。
A novel approach to nonviral DNA delivery is the use of combinations of DNA-binding proteins such as the yeast transcriptional activator GAL4 and plasmid DNA containing the specific binding sequence of the DNA-binding protein inserted within it, in addition to the gene of interest to be transferred into target cells. The amino terminal 147 amino acids of GAL4 contain a DNA-binding domain that has been shown to bind specifically to a 17 bp nucleotide recognition sequence, while the amino terminal 74 amino have been shown to be sufficient to target large heterologous proteins to the nucleus. Although if has been previously exploited as a gene transfer vehicle, the exact relationship between GAL4's DNA binding and nuclear targeting activities has not been investigated. Using gel mobility shift assays and ELISA-based binding assays, this study examines this issue directly, establishing the mutual exclusivity of the DNA-binding and nuclear targeting activities of GAL4. We demonstrate that GAL4(1-147) can specifically enhance transfection of plasmids containing the 17 bp recognition sequence. interestingly, we found that the nuclear localization signal (NLS) of GAL4 is district from conventional NLSs, such as those of the SV40 large tumor antigen and bipartite NLSs,in that it is recognized exclusively by the nuclear pore targeting p-subunit of the NLS-receptor importin complex, rather than the alpha-subunit. Specific binding to DNA was blocked by beta-subunit binding;, while the converse was also-true, making the GAL4-NLS novel in being regulated by DNA binding; this may:play an important role in effecting:release of GAL4 from the beta-subunit unit following transport through the nuclear pore. study encompasses the first direct analysis of NLS recognition/accessibility in vehicles for nonviral DNA transfer, with the results-having relevance to the use of GAL4 and comparable DNA-binding proteins in such vehicles.